Fetal liver CD34+ contain human immune and endothelial progenitors and mediate solid tumor rejection in NOG mice.

BACKGROUND: Transplantation of CD34+ hematopoietic stem and progenitor cells (HSPC) into immunodeficient mice is an established method to generate humanized mice harbouring a human immune system. Different sources and methods for CD34+ isolation have been employed by various research groups, resulting in customized models that are difficult to compare. A more detailed characterization of CD34+ isolates is needed for a better understanding of engraftable hematopoietic and potentially non-hematopoietic cells. Here we have performed a direct comparison of CD34+ isolated from cord blood (CB-CD34+) or fetal liver (FL-CD34+ and FL-CD34+CD14-) and their engraftment into immunocompromised NOD/Shi-scid Il2rgnull (NOG) mice. METHODS: NOG mice were transplanted with either CB-CD34+, FL-CD34+ or FL-CD34+CD14- to generate CB-NOG, FL-NOG and FL-CD14--NOG, respectively. After 15-20 weeks, the mice were sacrificed and human immune cell reconstitution was assessed in blood and several organs. Liver sections were pathologically assessed upon Haematoxylin and Eosin staining. To assess the capability of allogenic tumor rejection in CB- vs. FL-reconstituted mice, animals were subcutaneously engrafted with an HLA-mismatched melanoma cell line. Tumor growth was assessed by calliper measurements and a Luminex-based assay was used to compare the cytokine/chemokine profiles. RESULTS: We show that CB-CD34+ are a uniform population of HSPC that reconstitute NOG mice more rapidly than FL-CD34+ due to faster B cell development. However, upon long-term engraftment, FL-NOG display increased numbers of neutrophils, dendritic cells and macrophages in multiple tissues. In addition to HSPC, FL-CD34+ isolates contain non-hematopoietic CD14+ endothelial cells that enhance the engraftment of the human immune system in FL-NOG mice. We demonstrate that these CD14+CD34+ cells are capable of reconstituting Factor VIII-producing liver sinusoidal endothelial cells (LSEC) in FL-NOG. However, CD14+CD34+ also contribute to hepatic sinusoidal dilatation and immune cell infiltration, which may culminate in a graft-versus-host disease (GVHD) pathology upon long-term engraftment. Finally, using an HLA-A mismatched CDX melanoma model, we show that FL-NOG, but not CB-NOG, can mount a graft-versus-tumor (GVT) response resulting in tumor rejection. CONCLUSION: Our results highlight important phenotypical and functional differences between CB- and FL-NOG and reveal FL-NOG as a potential model to study hepatic sinusoidal dilatation and mechanisms of GVT.

The TREK-1 potassium channel is involved in both the analgesic and anti-proliferative effects of riluzole in bone cancer pain.

BACKGROUND: The metastasis of tumors into bone tissue typically leads to intractable pain that is both very disabling and particularly difficult to manage. We investigated here whether riluzole could have beneficial effects for the treatment of prostate cancer-induced bone pain and how it could influence the development of bone metastasis. METHODS: We used a bone pain model induced by intratibial injection of human PC3 prostate cancer cells into male SCID mice treated or not with riluzole administered in drinking water. We also used riluzole in vitro to assess its possible effect on PC3 cell viability and functionality, using patch-clamp. RESULTS: Riluzole had a significant preventive effect on both evoked and spontaneous pain involving the TREK-1 potassium channel. Riluzole did not interfere with PC3-induced bone loss or bone remodeling in vivo. It also significantly decreased PC3 cell viability in vitro. The antiproliferative effect of riluzole is correlated with a TREK-1-dependent membrane hyperpolarization in these cells. CONCLUSION: The present data suggest that riluzole could be very useful to manage evoked and spontaneous hypersensitivity in cancer-induced bone pain and has no significant adverse effect on cancer progression.

Shikonin blocks CAF-induced TNBC metastasis by suppressing mitochondrial biogenesis through GSK-3ß/NEDD4-1 mediated phosphorylation-dependent degradation of PGC-1α.

BACKGROUND: Triple-negative breast cancer (TNBC) is characterized by its high metastatic potential, which results in poor patient survival. Cancer-associated fibroblasts (CAFs) are crucial in facilitating TNBC metastasis via induction of mitochondrial biogenesis. However, how to inhibit CAF-conferred mitochondrial biogenesis is still needed to explore. METHODS: We investigated metastasis using wound healing and cell invasion assays, 3D-culture, anoikis detection, and NOD/SCID mice. Mitochondrial biogenesis was detected by MitoTracker green FM staining, quantification of mitochondrial DNA levels, and blue-native polyacrylamide gel electrophoresis. The expression, transcription, and phosphorylation of peroxisome-proliferator activated receptor coactivator 1α (PGC-1α) were detected by western blotting, chromatin immunoprecipitation, dual-luciferase reporter assay, quantitative polymerase chain reaction, immunoprecipitation, and liquid chromatography-tandem mass spectrometry. The prognostic role of PGC-1α in TNBC was evaluated using the Kaplan-Meier plotter database and clinical breast cancer tissue samples. RESULTS: We demonstrated that PGC-1α indicated lymph node metastasis, tumor thrombus formation, and poor survival in TNBC patients, and it was induced by CAFs, which functioned as an inducer of mitochondrial biogenesis and metastasis in TNBC. Shikonin impeded the CAF-induced PGC-1α expression, nuclear localization, and interaction with estrogen-related receptor alpha (ERRα), thereby inhibiting PGC-1α/ERRα-targeted mitochondrial genes. Mechanistically, the downregulation of PGC-1α was mediated by synthase kinase 3ß-induced phosphorylation of PGC-1α at Thr295, which associated with neural precursor cell expressed developmentally downregulated 4e1 recognition and subsequent degradation by ubiquitin proteolysis. Mutation of PGC-1α at Thr295 negated the suppressive effects of shikonin on CAF-stimulated TNBC mitochondrial biogenesis and metastasis in vitro and in vivo. CONCLUSIONS: Our findings indicate that PGC-1α is a viable target for blocking TNBC metastasis by disrupting mitochondrial biogenesis, and that shikonin merits potential for treatment of TNBC metastasis as an inhibitor of mitochondrial biogenesis through targeting PGC-1α.

20(R)-Panaxatriol enhances METTL3-mediated m6A modification of STUB1 to inhibit autophagy and exert antitumor effects in Triple-Negative Breast Cancer cells.

BACKGROUND: Aberrant activation of autophagy in triple-negative breast cancer (TNBC) has led researchers to investigate potential therapeutic strategies targeting this process. The regulation of autophagy is significantly influenced by METTL3. Our previous research has shown that the Panax ginseng-derived compound, 20(R)-panaxatriol (PT), has potential as an anti-tumor agent. However, it remains unclear whether PT can modulate autophagy through METTL3 to exert its anti-tumor effects. OBJECTIVE: Our objective is to investigate whether PT can regulate autophagy in TNBC cells and elucidate the molecular mechanisms. STUDY DESIGN: For in vitro experiments, we employed SUM-159-PT and MDA-MB-231 cells. While in vivo experiments involved BALB/c nude mice and NOD/SCID mice. METHODS: In vitro, TNBC cells were treated with PT, and cell lines with varying expression levels of METTL3 were established. We assessed the impact on tumor cell activity and autophagy by analyzing autophagic flux, Western Blot (WB), and methylation levels. In vivo, subcutaneous transplantation models were established in BALB/c nude and NOD/SCID mice to observe the effect of PT on TNBC growth. HE staining and immunofluorescence were employed to analyze histopathological changes in tumor tissues. MeRIP-seq and dual-luciferase reporter gene assays were used to identify key downstream targets. Additionally, the silencing of STIP1 Homology And U-Box Containing Protein 1 (STUB1) explored PT's effects. The mechanism of PT's action on STUB1 via METTL3 was elucidated through mRNA stability assays, mRNA alternative splicing analysis, and nuclear-cytoplasmic mRNA separation. RESULTS: In both in vivo and in vitro experiments, it was discovered that PT significantly upregulates the expression of METTL3, leading to autophagy inhibition and therapeutic effects in TNBC. Simultaneously, through MeRIP-seq analysis and dual-luciferase reporter gene assays, we have demonstrated that PT modulates STUB1 via METTL3, influencing autophagy in TNBC cells. Furthermore, intriguingly, PT extends the half-life of STUB1 mRNA by enhancing its methylation modification, thereby enhancing its stability. CONCLUSION: In summary, our research reveals that PT increases STUB1 m6A modification through a METTL3-mediated mechanism in TNBC cells, inhibiting autophagy and further accentuating its anti-tumor properties. Our study provides novel mechanistic insights into TNBC pathogenesis and potential drug targets for TNBC.

Development and Validation of a Simple and Reliable HPLC-UV Method for Determining Gemcitabine Levels: Application in Pharmacokinetic Analysis.

Background and Objectives: Gemcitabine has been used to treat various solid cancers, including, since 1997, metastatic pancreatic cancer. Here, we developed an HPLC-UV method to determine serum gemcitabine levels and use it in pharmacokinetic studies. Materials and Methods: The analysis was performed after a single protein precipitation step on a reversed-phase column, isocratically eluted with sodium phosphate buffer and methanol. For the pharmacokinetic study, NOD/SCID mice received a single dose of gemcitabine at 100 mg/kg by either subcutaneous (SC) or intraperitoneal (IP) administration. Blood samples were collected at 5, 15, and 30 min and 1, 2, 4, and 6 h after the administration of gemcitabine for further analysis. Results: The duration of the analysis was ~12.5 min. The calibration curve was linear (r2 = 0.999) over the range of 1-400 µM. The mean recovery of GEM was 96.53% and the limit of detection was 0.166 µΜ. T1/2, Tmax, Cmax, AUC0-t, and clearance were 64.49 min, 5.00 min, 264.88 µmol/L, 9351.95 µmol/L*min, and 0.0103(mg)/(µmol/L)/min, respectively, for the SC administration. The corresponding values for the IP administration were 59.34 min, 5.00 min, 300.73 µmol/L, 8981.35 µmol/L*min and 0.0108(mg)/(µmol/L)/min (not statistically different from the SC administration). Conclusions: A simple, valid, sensitive, and inexpensive method for the measurement of gemcitabine in serum has been developed. This method may be useful for monitoring gemcitabine levels in cancer patients as part of therapeutic drug monitoring.

Simultaneous targeting of Tim3 and A2a receptors modulates MSLN-CAR T cell antitumor function in a human cervical tumor xenograft model.

Introduction: Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of hematological malignancies. However, its efficacy in solid tumors is limited by the immunosuppressive tumor microenvironment that compromises CAR T cell antitumor function in clinical settings. To overcome this challenge, researchers have investigated the potential of inhibiting specific immune checkpoint receptors, including A2aR (Adenosine A2 Receptor) and Tim3 (T cell immunoglobulin and mucin domain-containing protein 3), to enhance CAR T cell function. In this study, we evaluated the impact of genetic targeting of Tim3 and A2a receptors on the antitumor function of human mesothelin-specific CAR T cells (MSLN-CAR) in vitro and in vivo. Methods: Second-generation anti-mesothelin CAR T cells were produced using standard cellular and molecular techniques. A2aR-knockdown and/or Tim3- knockdown anti-mesothelin-CAR T cells were generated using shRNA-mediated gene silencing. The antitumor function of CAR T cells was evaluated by measuring cytokine production, proliferation, and cytotoxicity in vitro through coculture with cervical cancer cells (HeLa cell line). To evaluate in vivo antitumor efficacy of manufactured CAR T cells, tumor growth and mouse survival were monitored in a human cervical cancer xenograft model. Results: In vitro experiments demonstrated that knockdown of A2aR alone or in combination with Tim3 significantly improved CAR T cell proliferation, cytokine production, and cytotoxicity in presence of tumor cells in an antigen-specific manner. Furthermore, in the humanized xenograft model, both double knockdown CAR T cells and control CAR T cells could effectively control tumor growth. However, single knockdown CAR T cells were associated with reduced survival in mice. Conclusion: These findings highlight the potential of concomitant genetic targeting of Tim3 and A2a receptors to augment the efficacy of CAR T cell therapy in solid tumors. Nevertheless, caution should be exercised in light of our observation of decreased survival in mice treated with single knockdown MSLN-CAR T cells, emphasizing the need for careful efficacy considerations.

Repositioning of antiarrhythmics for prostate cancer treatment: a novel strategy to reprogram cancer-associated fibroblasts towards a tumor-suppressive phenotype.

BACKGROUND: Cancer-associated fibroblasts (CAFs) play a significant role in fueling prostate cancer (PCa) progression by interacting with tumor cells. A previous gene expression analysis revealed that CAFs up-regulate genes coding for voltage-gated cation channels, as compared to normal prostate fibroblasts (NPFs). In this study, we explored the impact of antiarrhythmic drugs, known cation channel inhibitors, on the activated state of CAFs and their interaction with PCa cells. METHODS: The effect of antiarrhythmic treatment on CAF activated phenotype was assessed in terms of cell morphology and fibroblast activation markers. CAF contractility and migration were evaluated by 3D gel collagen contraction and scratch assays, respectively. The ability of antiarrhythmics to impair CAF-PCa cell interplay was investigated in CAF-PCa cell co-cultures by assessing tumor cell growth and expression of epithelial-to-mesenchymal transition (EMT) markers. The effect on in vivo tumor growth was assessed by subcutaneously injecting PCa cells in SCID mice and intratumorally administering the medium of antiarrhythmic-treated CAFs or in co-injection experiments, where antiarrhythmic-treated CAFs were co-injected with PCa cells. RESULTS: Activated fibroblasts show increased membrane conductance for potassium, sodium and calcium, consistently with the mRNA and protein content analysis. Antiarrhythmics modulate the expression of fibroblast activation markers. Although to a variable extent, these drugs also reduce CAF motility and hinder their ability to remodel the extracellular matrix, for example by reducing MMP-2 release. Furthermore, conditioned medium and co-culture experiments showed that antiarrhythmics can, at least in part, reverse the protumor effects exerted by CAFs on PCa cell growth and plasticity, both in androgen-sensitive and castration-resistant cell lines. Consistently, the transcriptome of antiarrhythmic-treated CAFs resembles that of tumor-suppressive NPFs. In vivo experiments confirmed that the conditioned medium or the direct coinjection of antiarrhythmic-treated CAFs reduced the tumor growth rate of PCa xenografts. CONCLUSIONS: Collectively, such data suggest a new therapeutic strategy for PCa based on the repositioning of antiarrhythmic drugs with the aim of normalizing CAF phenotype and creating a less permissive tumor microenvironment.

Breast cancer exploits neural signaling pathways for bone-to-meninges metastasis.

The molecular mechanisms that regulate breast cancer cell (BCC) metastasis and proliferation within the leptomeninges (LM) are poorly understood, which limits the development of effective therapies. In this work, we show that BCCs in mice can invade the LM by abluminal migration along blood vessels that connect vertebral or calvarial bone marrow and meninges, bypassing the blood-brain barrier. This process is dependent on BCC engagement with vascular basement membrane laminin through expression of the neuronal pathfinding molecule integrin α6. Once in the LM, BCCs colocalize with perivascular meningeal macrophages and induce their expression of the prosurvival neurotrophin glial-derived neurotrophic factor (GDNF). Intrathecal GDNF blockade, macrophage-specific GDNF ablation, or deletion of the GDNF receptor neural cell adhesion molecule (NCAM) from BCCs inhibits breast cancer growth within the LM. These data suggest integrin α6 and the GDNF signaling axis as new therapeutic targets against breast cancer LM metastasis.

BUB1 regulates non-homologous end joining pathway to mediate radioresistance in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer subtype often treated with radiotherapy (RT). Due to its intrinsic heterogeneity and lack of effective targets, it is crucial to identify novel molecular targets that would increase RT efficacy. Here we demonstrate the role of BUB1 (cell cycle Ser/Thr kinase) in TNBC radioresistance and offer a novel strategy to improve TNBC treatment. METHODS: Gene expression analysis was performed to look at genes upregulated in TNBC patient samples compared to other subtypes. Cell proliferation and clonogenic survivals assays determined the IC50 of BUB1 inhibitor (BAY1816032) and radiation enhancement ratio (rER) with pharmacologic and genomic BUB1 inhibition. Mammary fat pad xenografts experiments were performed in CB17/SCID. The mechanism through which BUB1 inhibitor sensitizes TNBC cells to radiotherapy was delineated by γ-H2AX foci assays, BLRR, Immunoblotting, qPCR, CHX chase, and cell fractionation assays. RESULTS: BUB1 is overexpressed in BC and its expression is considerably elevated in TNBC with poor survival outcomes. Pharmacological or genomic ablation of BUB1 sensitized multiple TNBC cell lines to cell killing by radiation, although breast epithelial cells showed no radiosensitization with BUB1 inhibition. Kinase function of BUB1 is mainly accountable for this radiosensitization phenotype. BUB1 ablation also led to radiosensitization in TNBC tumor xenografts with significantly increased tumor growth delay and overall survival. Mechanistically, BUB1 ablation inhibited the repair of radiation-induced DNA double strand breaks (DSBs). BUB1 ablation stabilized phospho-DNAPKcs (S2056) following RT such that half-lives could not be estimated. In contrast, RT alone caused BUB1 stabilization, but pre-treatment with BUB1 inhibitor prevented stabilization (t1/2, ~8 h). Nuclear and chromatin-enriched fractionations illustrated an increase in recruitment of phospho- and total-DNAPK, and KAP1 to chromatin indicating that BUB1 is indispensable in the activation and recruitment of non-homologous end joining (NHEJ) proteins to DSBs. Additionally, BUB1 staining of TNBC tissue microarrays demonstrated significant correlation of BUB1 protein expression with tumor grade. CONCLUSIONS: BUB1 ablation sensitizes TNBC cell lines and xenografts to RT and BUB1 mediated radiosensitization may occur through NHEJ. Together, these results highlight BUB1 as a novel molecular target for radiosensitization in women with TNBC.

KSHV infection of B cells primes protective T cell responses in humanized mice.

Kaposi sarcoma associated herpesvirus (KSHV) is associated with around 1% of all human tumors, including the B cell malignancy primary effusion lymphoma (PEL), in which co-infection with the Epstein Barr virus (EBV) can almost always be found in malignant cells. Here, we demonstrate that KSHV/EBV co-infection of mice with reconstituted human immune systems (humanized mice) leads to IgM responses against both latent and lytic KSHV antigens, and expansion of central and effector memory CD4+ and CD8+ T cells. Among these, KSHV/EBV dual-infection allows for the priming of CD8+ T cells that are specific for the lytic KSHV antigen K6 and able to kill KSHV/EBV infected B cells. This suggests that K6 may represent a vaccine antigen for the control of KSHV and its associated pathologies in high seroprevalence regions, such as Sub-Saharan Africa.

Development of a novel rodent model for dog heartworm microfilaremia using the severe-combined immunodeficiency mouse.

Dirofilaria immitis is a mosquito-borne parasitic nematode that causes fatal heartworm disease in canids. The microfilariae are essential for research, including drug screening and mosquito-parasite interactions. However, no reliable methods for maintaining microfilaria long-term are currently available. Therefore, we used severe combined immunodeficiency (SCID) mice to develop a reliable method for maintaining D. immitis microfilaria. SCID mice were injected intravenously with microfilariae isolated from a D. immitis-infected dog. Microfilariae were detected in blood collected from the tail vein 218 days post-inoculation (dpi) and via cardiac puncture 296 dpi. Microfilariae maintained in and extracted from SCID mice showed infectivity and matured into third-stage larvae (L3s) in the vector mosquito Aedes aegypti. L3s can develop into the fourth stage larvae in vitro. Microfilariae from SCID mice respond normally to ivermectin in vitro. The microfilariae in SCID mice displayed periodicity in the peripheral circulation. The SCID mouse model aided in the separation of microfilariae from cryopreserved specimens. The use of SCID mice enabled the isolation and sustained cultivation of microfilariae from clinical samples. These findings highlight the usefulness of the SCID mouse model for studying D. immitis microfilaremia in canine heartworm research.

Bone Marrow Mesenchymal Stem Cells Promote Ovarian Cancer Cell Proliferation via Cytokine Interactions.

Bone marrow mesenchymal stem cells (BMSCs) are key players in promoting ovarian cancer cell proliferation, orchestrated by the dynamic interplay between cytokines and their interactions with immune cells; however, the intricate crosstalk among BMSCs and cytokines has not yet been elucidated. Here, we aimed to investigate interactions between BMSCs and ovarian cancer cells. We established BMSCs with a characterized morphology, surface marker expression, and tri-lineage differentiation potential. Ovarian cancer cells (SKOV3) cultured with conditioned medium from BMSCs showed increased migration, invasion, and colony formation, indicating the role of the tumor microenvironment in influencing cancer cell behavior. BMSCs promoted SKOV3 tumorigenesis in nonobese diabetic/severe combined immunodeficiency mice, increasing tumor growth. The co-injection of BMSCs increased the phosphorylation of p38 MAPK and GSK-3ß in SKOV3 tumors. Co-culturing SKOV3 cells with BMSCs led to an increase in the expression of cytokines, especially MCP-1 and IL-6. These findings highlight the influence of BMSCs on ovarian cancer cell behavior and the potential involvement of specific cytokines in mediating these effects. Understanding these mechanisms will highlight potential therapeutic avenues that may halt ovarian cancer progression.

Targeting the long non-coding RNA MIAT for the treatment of fibroids in an animal model.

Our previous studies indicated that there is overexpression of MIAT in fibroids and MIAT is a sponge for the miR-29 family in these tumors. The objective of the present study was to determine if the knockdown of MIAT in fibroid xenografts will increase miR-29 levels and reduce the expression of genes targeted by this miRNA such as collagen and cell cycle regulatory proteins in a mouse model for fibroids. Ovariectomized CB-17 SCID/Beige mice bearing estrogen/progesterone pellets were implanted subcutaneously in the flank with equal weight of fibroid explants which had been transduced by lentivirus for either control (empty vector) or MIAT knockdown for four weeks (n=7). Knockdown of MIAT in fibroid xenografts resulted in a 30% reduction of tumor weight and a marked increase in miR-29a, -b, and -c levels in the xenografts. There was reduced cell proliferation and expression of cell cycle regulatory genes CCND1, CDK2, and E2F1 and no significant changes in apoptosis. The xenografts with MIAT knockdown expressed lower mRNA and protein levels of FN1, COL3A1, and TGF-ß3, and total collagen protein. Targeting MIAT, which sponges the pro-fibrotic miR-29 family, is an effective therapy for fibroids by reducing cell proliferation and thereby, tumor growth and accumulation of ECM, which is a hallmark of these benign gynecologic tumors.

S309-CAR-NK cells bind the Omicron variants in vitro and reduce SARS-CoV-2 viral loads in humanized ACE2-NSG mice.

Recent progress on chimeric antigen receptor (CAR)-NK cells has shown promising results in treating CD19-positive lymphoid tumors with minimal toxicities [including graft versus host disease (GvHD) and cytokine release syndrome (CRS) in clinical trials. Nevertheless, the use of CAR-NK cells in combating viral infections has not yet been fully explored. Previous studies have shown that CAR-NK cells expressing S309 single-chain fragment variable (scFv), hereinafter S309-CAR-NK cells, can bind to SARS-CoV-2 wildtype pseudotyped virus (PV) and effectively kill cells expressing wild-type spike protein in vitro. In this study, we further demonstrate that the S309-CAR-NK cells can bind to different SARS-CoV-2 variants, including the B.1.617.2 (Delta), B.1.621 (Mu), and B.1.1.529 (Omicron) variants in vitro. We also show that S309-CAR-NK cells reduce virus loads in the NOD/SCID gamma (NSG) mice expressing the human angiotensin-converting enzyme 2 (hACE2) receptor challenged with SARS-CoV-2 wild-type (strain USA/WA1/2020). Our study demonstrates the potential use of S309-CAR-NK cells for inhibiting infection by SARS-CoV-2 and for the potential treatment of COVID-19 patients unresponsive to otherwise currently available therapeutics. IMPORTANCE: Chimeric antigen receptor (CAR)-NK cells can be "off-the-shelf" products that treat various diseases, including cancer, infections, and autoimmune diseases. In this study, we engineered natural killer (NK) cells to express S309 single-chain fragment variable (scFv), to target the Spike protein of SARS-CoV-2, hereinafter S309-CAR-NK cells. Our study shows that S309-CAR-NK cells are effective against different SARS-CoV-2 variants, including the B.1.617.2 (Delta), B.1.621 (Mu), and B.1.1.529 (Omicron) variants. The S309-CAR-NK cells can (i) directly bind to SARS-CoV-2 pseudotyped virus (PV), (ii) competitively bind to SARS-CoV-2 PV with 293T cells expressing the human angiotensin-converting enzyme 2 (hACE2) receptor (293T-hACE2 cells), (iii) specifically target and lyse A549 cells expressing the spike protein, and (iv) significantly reduce the viral loads of SARS-CoV-2 wild-type (strain USA/WA1/2020) in the lungs of NOD/SCID gamma (NSG) mice expressing hACE2 (hACE2-NSG mice). Altogether, the current study demonstrates the potential use of S309-CAR-NK immunotherapy as an alternative treatment for COVID-19 patients.

[52Mn]Mn-BPPA-Trastuzumab: A Promising HER2-Specific PET Radiotracer.

This study aimed to develop a novel radiotracer using trastuzumab and the long-lived [52Mn]Mn isotope for HER2-targeted therapy selection and monitoring. A new Mn(II) chelator, BPPA, synthesized from a rigid bispyclen platform possessing a picolinate pendant arm, formed a stable and inert Mn(II) complex with favorable relaxation properties. BPPA was converted into a bifunctional chelator (BFC), conjugated to trastuzumab, and labeled with [52Mn]Mn isotope. In comparison to DOTA-GA-trastuzumab, the BPPA-trastuzumab conjugate exhibits a labeling efficiency with [52Mn]Mn approximately 2 orders of magnitude higher. In female CB17 SCID mice bearing 4T1 (HER2-) and MDA-MB-HER2+ (HER2+) xenografts, [52Mn]Mn-BPPA-trastuzumab demonstrated superior uptake in HER2+ cells on day 3, with a 3-4 fold difference observed on day 7. Overall, the hexadentate BPPA chelator proves to be exceptional in binding Mn(II). Upon coupling with trastuzumab as a BFC ligand, it becomes an excellent imaging probe for HER2-positive tumors. [52Mn]Mn-BPPA-trastuzumab enables an extended imaging time window and earlier detection of HER2-positive tumors with superior tumor-to-background contrast.

A novel humanized mouse model for HIV and tuberculosis co-infection studies.

Background: Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), continues to be a major public health problem worldwide. The human immunodeficiency virus (HIV) is another equally important life-threatening pathogen. HIV infection decreases CD4+ T cell levels markedly increasing Mtb co-infections. An appropriate animal model for HIV/Mtb co-infection that can recapitulate the diversity of the immune response in humans during co-infection would facilitate basic and translational research in HIV/Mtb infections. Herein, we describe a novel humanized mouse model. Methods: The irradiated NSG-SGM3 mice were transplanted with human CD34+ hematopoietic stem cells, and the humanization was monitored by staining various immune cell markers for flow cytometry. They were challenged with HIV and/or Mtb, and the CD4+ T cell depletion and HIV viral load were monitored over time. Before necropsy, the live mice were subjected to pulmonary function test and CT scan, and after sacrifice, the lung and spleen homogenates were used to determine Mtb load (CFU) and cytokine/chemokine levels by multiplex assay, and lung sections were analyzed for histopathology. The mouse sera were subjected to metabolomics analysis. Results: Our humanized NSG-SGM3 mice were able to engraft human CD34+ stem cells, which then differentiated into a full-lineage of human immune cell subsets. After co-infection with HIV and Mtb, these mice showed decrease in CD4+ T cell counts overtime and elevated HIV load in the sera, similar to the infection pattern of humans. Additionally, Mtb caused infections in both lungs and spleen, and induced granulomatous lesions in the lungs. Distinct metabolomic profiles were also observed in the tissues from different mouse groups after co-infections. Conclusion: The humanized NSG-SGM3 mice are able to recapitulate the pathogenic effects of HIV and Mtb infections and co-infection at the pathological, immunological and metabolism levels and are therefore a reproducible small animal model for studying HIV/Mtb co-infection.

Shikonin reduces M2 macrophage population in ovarian cancer by repressing exosome production and the exosomal galectin 3-mediated ß-catenin activation.

BACKGROUND: Shikonin (SK), a naphthoquinone with anti-tumor effects, has been found to decrease production of tumor-associated exosomes (exo). This study aims to verify the treatment effect of SK on ovarian cancer (OC) cells, especially on the production of exo and their subsequent effect on macrophage polarization. METHODS: OC cells SKOV3 and A2780 were treated with SK. The exo were isolated from OC cells with or without SK treatment, termed OC exo and SK OC exo, respectively. These exo were used to treat PMA-induced THP-1 cells (M0 macrophages). M2 polarization of macrophages was determined by measuring the M2 specific cell surface markers CD163 and CD206 as well as the secretion of M2 cytokine IL-10. The functions of galectin 3 (LGALS3/GAL3) and ß-catenin in macrophage polarization were determined by gain- or loss-of-function assays. CB-17 SCID mice were subcutaneously injected with SKOV3 cells to generate xenograft tumors, followed by OC exo or SK OC exo treatment for in vivo experiments. RESULTS: SK suppressed viability, migration and invasion, and apoptosis resistance of OC cells in vitro. Compared to OC exo, SK OC exo reduced the M2 polarization of macrophages. Regarding the mechanism, SK reduced exo production in cancer cells, and it decreased the protein level of GAL3 in exo and recipient macrophages, leading to decreased ß-catenin activation. M2 polarization of macrophages was restored by LGALS3 overexpression but decreased again by the ß-catenin inhibitor FH535. Compared to OC exo, the SK OC exo treatment reduced the xenograft tumor growth in mice, and it decreased the M2 macrophage infiltration within tumor tissues. CONCLUSION: This study suggests that SK reduces M2 macrophage population in OC by repressing exo production and blocking exosomal GAL3-mediated ß-catenin activation.

Spatial distribution of tumor-associated macrophages in an orthotopic prostate cancer mouse model.

Mounting evidence suggests that the immune landscape within prostate tumors influences progression, metastasis, treatment response, and patient outcomes. In this study, we investigated the spatial density of innate immune cell populations within NOD.SCID orthotopic prostate cancer xenografts following microinjection of human DU145 prostate cancer cells. Our laboratory has previously developed nanoscale liposomes that attach to leukocytes via conjugated E-selectin (ES) and kill cancer cells via TNF-related apoptosis inducing ligand (TRAIL). Immunohistochemistry (IHC) staining was performed on tumor samples to identify and quantify leukocyte infiltration for different periods of tumor growth and E-selectin/TRAIL (EST) liposome treatments. We examined the spatial-temporal dynamics of three different immune cell types infiltrating tumors using QuPath image analysis software. IHC staining revealed that F4/80+ tumor-associated macrophages (TAMs) were the most abundant immune cells in all groups, irrespective of time or treatment. The density of TAMs decreased over the course of tumor growth and decreased in response to EST liposome treatments. Intratumoral versus marginal analysis showed a greater presence of TAMs in the marginal regions at 3 weeks of tumor growth which became more evenly distributed over time and in tumors treated with EST liposomes. TUNEL staining indicated that EST liposomes significantly increased cell apoptosis in treated tumors. Additionally, confocal microscopy identified liposome-coated TAMs in both the core and periphery of tumors, highlighting the ability of liposomes to infiltrate tumors by "piggybacking" on macrophages. The results of this study indicate that TAMs represent the majority of innate immune cells within NOD.SCID orthotopic prostate tumors, and spatial density varies widely as a function of tumor size, duration of tumor growth, and treatment of EST liposomes.

Representation of genomic intratumor heterogeneity in multi-region non-small cell lung cancer patient-derived xenograft models.

Patient-derived xenograft (PDX) models are widely used in cancer research. To investigate the genomic fidelity of non-small cell lung cancer PDX models, we established 48 PDX models from 22 patients enrolled in the TRACERx study. Multi-region tumor sampling increased successful PDX engraftment and most models were histologically similar to their parent tumor. Whole-exome sequencing enabled comparison of tumors and PDX models and we provide an adapted mouse reference genome for improved removal of NOD scid gamma (NSG) mouse-derived reads from sequencing data. PDX model establishment caused a genomic bottleneck, with models often representing a single tumor subclone. While distinct tumor subclones were represented in independent models from the same tumor, individual PDX models did not fully recapitulate intratumor heterogeneity. On-going genomic evolution in mice contributed modestly to the genomic distance between tumors and PDX models. Our study highlights the importance of considering primary tumor heterogeneity when using PDX models and emphasizes the benefit of comprehensive tumor sampling.

BET inhibition decreases HMGCS2 and sensitizes resistant pancreatic tumors to gemcitabine.

Efforts to develop targetable molecular bases for drug resistance for pancreatic ductal adenocarcinoma (PDAC) have been equivocally successful. Using RNA-seq and ingenuity pathway analysis we identified that the superpathway of cholesterol biosynthesis is upregulated in gemcitabine resistant (gemR) tumors using a unique PDAC PDX model with resistance to gemcitabine acquired in vivo. Analysis of additional in vitro and in vivo gemR PDAC models showed that HMG-CoA synthase 2 (HMGCS2), an enzyme involved in cholesterol biosynthesis and rate limiting in ketogenesis, is overexpressed in these models. Mechanistic data demonstrate the novel findings that HMGCS2 contributes to gemR and confers metastatic properties in PDAC models, and that HMGCS2 is BRD4 dependent. Further, BET inhibitor JQ1 decreases levels of HMGCS2, sensitizes PDAC cells to gemcitabine, and a combination of gemcitabine and JQ1 induced regressions of gemR tumors in vivo. Our data suggest that decreasing HMGCS2 may reverse gemR, and that HMGCS2 represents a useful therapeutic target for treating gemcitabine resistant PDAC.